HIV-1 infection and AIDS continues to be a worldwide problem, even in the face of new combinations of highly active antiretroviral therapies. This research program continues to focus upon the development of novel RNA based inhibitors of HIV-1 replication for use in anti-HIV-1 gene therapy applications. RNA interference has proven to be a remarkably fascinating compilation of cellular mechanisms that regulate gene expression from the transcriptional through post-transcriptonal levels. With the potency of small interfering RNAs still greatly exceeding that of any single ribozyme, there has been a major emphasis shift in this program from ribozymes to RNA interference as an antiviral agent. Our goals are to enhance the functioning of anti-HIV siRNAs, and to elucidate the mechanism of action of effective siRNAs or micro RNAs with the goals of developing non-toxic anti-HIV siRNAs that minimize viral escape mutants and provide long-term inhibition of viral replication. Despite the growing and realistic excitement over RNAi, ribozymes can still perform useful functions. Recent findings emulate the native catalytic domain of plant viroids and virusoids to achieve more efficient ribozymes under physiological conditions. We have exploited this finding to provide the first demonstration that the native structure of the hairpin domain in plant viroids and virusoids can be applied to trans-cleaving ribozymes. This program will continue to exploit hammerhead ribozymes by incorporating this native conformation into the design of our anti-HIV ribozymes and continue to study the most effective ways of expressing these more highly active ribozyme motifs in hematopoietic cells. We have recently identified a new version of a potent anti-HIV-1 nucleolar localizing ribozyme from a ribozyme library. We are now in a position to design new ribozyme expression constructs that combine the most recent structural constraints with nucleolar trafficking ribozymes. In vivo testing of the most promising anti-HIV constructs will be carried out via collaboration with Dr. Ramesh Akkina at Colorado State Univ. He has established both a standard SCID-hu and the Rag2-/-yc-\- double knockout mouse models to study hematopoietic stem cell differentiation. In accordance with the changes that have taken place in this research program over the past four years, the specific aims have been adjusted accordingly. The revised aims are:Aim 1) Intracellular expression and functional analyses of anti-HIV shRNAs and micro RNAs: Aim 2) Intracellular expression, mechanism of action, and anti-HIV-1 studies of nucleolar localizing ribozymes and new ribozyme catalytic domain motifs: Aim 3) Functional analyses and differentiation potentials of anti-HIV RNA expressing hematopoietic cells in vitro and in vivo. The overall goal of this program remains that of developing potent RNA based anti-HIV inhibitory agents for use in human gene therapy treatment of HIV infection.